Methods for determining ligand binding to a target protein using a thermal shift assay

ABSTRACT

The invention is directed to a method of determining whether a non-purified sample contains a target protein bound to a ligand of interest comprising the steps of: a) exposing said non-purified sample to a temperature which is capable of causing or enhancing precipitation of the unbound target protein to a greater extent than it is capable of causing or enhancing precipitation of the target protein bound to said ligand; b) processing the product of step a) in order to separate soluble from insoluble protein; and c) analyzing either or both the soluble and insoluble protein fractions of step b) for the presence of target protein, wherein said target protein is not detected on the basis of enzymatic activity of a tag, peptide, polypeptide or protein fused thereto. Particularly, the invention may be used to determine whether drugs can bind to their protein targets in samples derived from patients to ascertain whether a certain drug can be used in a therapy for that patient. Additionally, the invention is directed to an instrument for use in the methods of the invention and use of a kit in the methods of the invention comprising an antibody and/or a non-protein fusion tag.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.14/057,920, filed Oct. 18, 2013, which is a continuation ofInternational Patent Application No. PCT/GB2012/050853, filed Apr. 18,2012, which claims the benefit of UK Patent Application No. GB1106548.9, filed Apr. 18, 2011, the entire contents of which are herebyincorporated by reference.

The present invention relates to methods of investigating protein ligandbinding interactions, in particular through the use of thermal shiftanalysis.

More particularly, the invention relates to methods for determiningligand binding to a non-purified target protein comprising steps ofheating the non-purified target protein and ligand and analysing theproduct to detect soluble target protein. In certain embodiments, themethods of the invention use a separation step to separate soluble frominsoluble proteins after heat treatment to estimate the amount ofsoluble target protein and thus thermally stable ligand bound targetprotein. The invention also relates to an instrument for use in themethods comprising a heating means, a means for separating soluble frominsoluble protein and a means for analysing soluble or insoluble proteinfor the presence of target protein. The use of a kit comprising anantibody or a non-protein fusion tag in the methods of the invention isalso described.

The detection of ligand binding to proteins is important in manydifferent areas of biology and medicine. Particularly, during thedevelopment of chemical compounds into drugs, it is important to know ifthe compound interacts with the drug target. The monitoring of targetprotein-ligand interactions can therefore be used in initial screeningfor interacting ligands from large chemical libraries, as well as duringoptimization of an initial ligand into a candidate drug. Further, it isimportant to understand the interaction of a drug with other proteins(so called “off target interactions”) where such interactions may resultin side effects of treatments.

In other medical applications, it is important to determine whether aparticular drug is able to bind its target protein in a patient or ananimal model (for the disease). For a drug to be efficient, it needs tobe absorbed in the stomach/gut (or if injected, it should enter theblood) and be transported to the right location in the body. If the drugis not targeted to an extracellular protein or receptor, the drug alsoneeds to be transported into the cell in order to allow it to access thetarget protein. During all these transport processes, the drug needs tobe stable and to avoid excretion from the kidney and degradation, e.g.in the liver or by cellular metabolic enzymes. The drug further needs tosurvive cellular drug resistance processes, such as degradation by P450enzymes or translocation by multi-drug efflux channels. Finally, thedrug needs to be able to bind to the drug target protein. Drugresistance in cancer and infection therapy is sometimes due to subtlemutations in the region of the drug-binding site on the target proteins.However, in the path from drug to target, drugs will meet many differentenvironments of the body and can potentially interact with manydifferent proteins along the way.

The high complexity of the path for the drug before it reaches thebinding site on the target protein is probably one reason why currentpredictive methods based on clinical diagnostics, expression profilingand sequencing only have limited success in predicting therapeuticefficiency. A potential means for measuring whether a drug has reachedits target is to perform direct measurement of the drug-target proteininteraction in the target cells of the body. Although this would notmeasure events downstream of the drug target, it would integrate allsteps from drug to target as described above. Such measurements maytherefore encompass many of the critical steps of therapeutic efficiencyand would be a valuable predictor of the efficiency of many drugs andtherefore as a clinical diagnostic tool. Thus, it is desirable to beable to detect ligand-protein interactions in non-purified samples e.g.those from patients, to study drug interactions and efficiencies.

Thermal shift assays have been developed in the art which can assessprotein-ligand binding where the protein is in purified form. Theseassays have been developed on the basis of two principles, namely that apurified protein will melt and unfold at a particular temperature andthat the binding of a ligand to a protein will thermally stabilise theprotein. Thus, the binding of a ligand to a protein can be detected onthe basis that the purified protein will show an increase in thermalstability once a ligand is bound and hence the protein will melt at ahigher temperature once ligand is bound than purified protein alone.Vedadi et al (PNAS, 103(43), 15835-15840, 2006) evaluated chemicalscreening methods to identify ligands that promote protein stability,crystallisation and structure determination. In these methods, thethermal stability of recombinant purified proteins was assessed afterscreening against small molecule libraries. An increase in proteinthermal stability and thus ligand binding was measured using eitherfluorimetry (where fluorescent probes were used) or static lightscattering. However, as discussed above, this method used purifiedproteins which melted at a particular temperature (as determined by areference sample using unligated protein) allowing an increase instability at the melting temperature to be measured.

Moreau et al. (Mol. BioSyst., 6, 1285-1292, 2010), recently used GFP asa reporter system to determine the stability of a target protein and itsligand associated stabilisation, where GFP was fused to the targetprotein. However, this method is not ideal. Firstly, the method requiresthe construction and expressions of a fusion protein and can thereforenot be used in natural cells and tissues but only in transformed cells.Further, the method can only be used to detect ligand binding toproteins which are less stable than GFP. Finally, the use of additivesor salts affects the stability of GFP and thus a control GFP must beused for every experiment.

Thus, the thermal shift assays described in the prior art were only usedin connection with purified protein, or in one case (Moreau et ai,supra) in connection with a purified protein mixed with one otherprotein after purification where the protein is fused to GFP. Incontrast to this, the inventors have developed an assay which can beused to determine binding of a ligand to a non-purified protein wherethe non-purified protein is not detected based on the enzymatic activityof any tag or peptide, polypeptide or protein fused thereto. Thus, theinventors have shown that it is possible for a non-purified protein e.g.in a cell, cell lysate or other complex liquid containing many differentbiomolecules, to unfold and precipitate with a characteristictemperature dependence, in a similar way to a purified protein. Thisdiscovery was unexpected since the conditions that are present in cellsand in nonpurified samples are quite different to the ones in a purifiedsample. Thus, in a non-purified sample or in a cell, it would beexpected that several different processes may affect the solubility of aprotein, which would act in parallel, such as different protein crowdingeffects or different chaperon or membrane interactions of partiallyunfolded proteins. The inventors used this finding as discussed above todevelop a thermal shift assay, which can detect ligand binding toproteins in non-purified samples, based on the ability of non-purifiedproteins to melt at characteristic temperatures. The method is genericand can be used for most target protein and ligand combination, unlikethe methods of the prior art. The assay investigates the thermalstability of the target protein in non-purified form at a particulartemperature where an increase in thermal stability is indicative ofligand binding. Thus, the thermal stability of the non-purified targetprotein with added ligand is compared to the thermal stability of thenon-purified target protein without ligand. Any increase in thermalstability of the non-purified target protein plus ligand compared tononpurified target protein without ligand indicates that ligand is boundto the nonpurified target protein. Particularly, any increase in thermalstability is determined by detecting whether or not the target proteinis soluble after heat treatment. The assay of the invention thereforeemploys a simple step of separating soluble from insoluble proteins toidentify any soluble target proteins. As discussed above such solubleproteins are associated with being thermally stable at the temperatureapplied to the sample and thus with having bound ligand. The separationstep to discriminate between soluble and insoluble proteins thus allowsthe assay of the invention to be used to detect any target protein andtherefore provides a generic method.

Therefore, in one aspect the invention provides a method for identifyinga ligand which is capable of binding to a target protein wherein saidtarget protein is non-purified comprising the steps of

(a) exposing a sample comprising said non-purified target protein and atest molecule, to a temperature which is capable of causing or enhancingprecipitation of said target protein

(b) processing the product of step (a) in order to separate soluble frominsoluble protein and

(c) analysing the soluble proteins of step (b) for the presence oftarget protein, wherein said target protein is not detected on the basisof enzymatic activity of a tag, peptide, polypeptide or protein fusedthereto.

Thus, as discussed above, the method of the invention is concerned withdetecting ligand binding to a target protein in a non-purified samplewhere surprisingly a target protein in such a sample is capable ofmelting or unfolding with a characteristic temperature. When a targetprotein is bound to a ligand, the thermal stability of the targetprotein is generally increased and thus the target protein may melt at ahigher temperature when ligand is bound than when no ligand is present.Applying a temperature to a sample which usually melts/unfolds unboundtarget protein may therefore result in unbound target protein beingunfolded and target protein to which a ligand is bound remaining foldedto a larger extent. The detection of higher levels of folded targetprotein is therefore indicative of ligand binding. Folded targetproteins are generally soluble whereas unfolded proteins are generallyinsoluble. Hence, the solubility of a protein is linked to its thermalstability. Thus, the detection of higher levels of soluble targetprotein after heat treatment using a temperature at which the targetprotein usually start to precipitate and become insoluble indicates thepresence of folded target protein with an increased thermal stabilityand hence ligand binding.

The invention is concerned with analysis of impure samples. This allowsthe technology to be used in a “biosensor” type method. Non-purifiedsamples, in particular clinical or environmental samples, which maycontain a ligand of interest can be analysed by adding the targetprotein to the sample, either as a purified protein or in a non-purifiedsample (e.g. as a cell lysate). Such methods allow quantification of thepresence of a drug or other analyte in a serum sample, even though thetarget protein is originally not present in the serum. A cell lysatecontaining the target protein, e.g. from the target cells of the drugcan be added to the clinical sample.

Thus, the present invention provides a more general method ofdetermining whether a non-purified sample contains a target proteinbound to a ligand of interest comprising the steps of:

(a) exposing said non-purified sample to a temperature which is capableof causing or enhancing precipitation of the unbound target protein to agreater extent than it is capable of causing or enhancing precipitationof the target protein bound to said ligand;(b) processing the product of step a) in order to separate soluble frominsoluble protein; and(c) analysing either or both the soluble and insoluble protein fractionsof step b) for the presence of target protein, wherein said targetprotein is not detected on the basis of enzymatic activity of a tag,peptide, polypeptide or protein fused thereto.

The skilled man is familiar with thermal shift analysis of purifiedproteins and the melting point curves produced thereby. The midpoint ofthe melting point curve may be taken to be the melting point of theprotein and this temperature can change on ligand binding. It isappreciated that depending on the nature of the shift caused by ligandbinding, at certain temperatures there may be some melting(precipitation) of both bound and unbound proteins but thatprecipitation occurs to a greater extent with the unbound protein. Thetemperatures at which the shift is visible and the amount ofprecipitated protein differs are discriminatory temperatures andtemperatures within that range can be used as a single discriminatorytemperature according to step (a) above. That is particularly so whenthe m.p. of unbound and bound target proteins are known and the methodis performed as an assay for the presence of target protein and/orligand in the sample. Thus ligand may be added to a sample to confirmthe presence of target protein.

The method of the invention is a generic method for determining ligandtarget protein binding in a non-purified sample. Unless otherwise clearfrom the context, discussion herein of “non-purified protein” appliesmutatis mutandis to ‘non-purified samples’. This method has manyadvantages over the methods of the art. Firstly, it abrogates the needto purify proteins in order to investigate ligand binding. Further, themethod does not require the recombinant expression of target protein orthe production of a protein containing a fusion reporter protein (suchas in Moreau et al., supra). It further allows the investigation ofligand binding in cell culture, animal or patient samples which was notpreviously possible using thermal shift binding assays. As discussedabove, this is important for analysing whether a particular drug can beefficiently used to treat disease in a particular patient and to assistin determining optimal dosage of the drug. For example, this hasimportant ramifications for the treatment of cancer and infectiousdiseases, where drug resistance often can occur. In such cases beingable to detect patients who would not be effectively treated with thedrug, allows other therapies to be commenced, or drug dosage to beadjusted.

Further, the detection steps of the method of the invention do notrequire the use of expensive equipment or machinery; indeed, theseparation step can be achieved using a filter and target proteindetected for example using antibodies. The method can be used for anyprotein to detect the binding of a ligand; there is no requirement todesign specific probes etc for each protein to be detected in themethod. Thus, the method of the invention represents an efficient,reliable way of determining protein-ligand binding in a non-purifiedsample. Additionally, as discussed further below, the method can beeasily multiplexed and used to screen libraries of ligands or proteinsfor interaction.

The term “target protein” as used herein, refers to a protein which isbeing assessed in the method of the invention for ligand binding. Thetarget protein can therefore be any protein which is present in asample. The target protein may be naturally occurring e.g. in a cell orcell lysate or animal or patient sample or may be recombinantlyexpressed e.g. may be expressed from a plasmid which has beentransformed into a cell. As mentioned above, the target protein may notinitially be present in the sample but may be added thereto toinvestigate the presence of ligand in the starting sample. Thus,according to the present invention, the ‘sample’ is the test samplewhich is treated in step (a) and this may be different from the startingsample, e.g. the clinical sample. Likewise, ligand may be added to thestarting sample. Additions of known amounts of target protein or ligandmay assist in obtaining quantitative data.

The target protein may be in wildtype form i.e. as it usually occurs innature or may comprise one or more mutations. Thus genes/cDNA/codingregions encoding a protein can be mutated to produce variants of thatprotein e.g. mutants with varying abilities to bind the ligand. Asdiscussed further below, these mutants can be produced in an expressionsystem wherein the variants, which for example have increased ligandbinding, can be selected using the methods of the invention.

Typically, the target protein will have a native or native-likeconformation and will be soluble. Native or native-like proteins areexpressed in soluble form and/or correctly folded. Native-like membraneproteins do not have to be present free in solution, but may be presentin cellular membranes or membrane vesicles rather than inclusion bodies.Thus native-like proteins are generally not insoluble, present ininclusion bodies, aggregated or misfolded.

The target protein may exist in the form of numerous variants across ananimal population. These variants may exist within a healthy animalpopulation, or the variation in the protein may lead to disease or drugresistance within a population. The methods of the invention provide ameans of screening a ligand across a range of different target proteinvariants. Such information may be useful in order to develop ligandsthat bind to certain protein variants specifically, or to determinewhich form of therapy may be most adequate for a patient based on theprotein variant which they naturally express. Thus, the method may berepeated with two or more target proteins, those target proteins beingvariants of the same protein.

A “soluble protein” can be defined in reference to possession of anative or native-like conformation. Further, a soluble protein can bedescribed as a protein which remains in the supernatant aftercentrifugation of a sample (with a prior lysis step if said protein iswithin a cell. Centrifugation can typically be carried out between 100 gand 20000 g. The duration of centrifugation can be from 1 minute(typically at least 10 minutes) to at least 1 hour, where the durationrequired generally decreases as the centrifugal force increases.Particularly suitable conditions for providing only soluble proteins inthe resultant supernatant include 30 minutes at 3000 g or 15 minutes at20000 g.

The term “non-purified target protein” refers to the target protein whennot in isolated form or alternatively viewed when present with othercompounds e.g. proteins. The non-purified target protein to be used inthe methods of the invention are in non-purified form before theaddition of the test molecule (potential ligand) or in the absence ofthe test molecule. Thus, the non-purified target protein is present withcompounds other than the test molecule (potential ligand) which istested to determine whether or not it is a ligand for the targetprotein. The non-purified target protein thus includes target proteinwhen comprised within or on cells, cell Iysates and samples obtaineddirectly from patients (human patients or animal patients or diseasemodels e.g. dog, cat, monkey, rabbit, mouse, rat etc) such as tissuesamples, blood, serum, plasma, lymph etc. The non-purified targetprotein includes target protein when comprised in one or more cellcolonies, where a cell colony is defined as a circumscribed group ofcells, normally derived from a single cell or small cluster of cellsgrowing on a solid or semi-solid medium (i.e. culture media with theaddition of 0.1% or greater agar). The non-purified target protein mayalso be comprised in a liquid culture of cells. A liquid culture ofcells may comprise cells which have all originated from a single celli.e. the cells within the liquid culture may be clonal, or the liquidculture may comprise a suspension of different cells. The cells of thecolonies or in liquid culture may be prokaryotic i.e. bacteria oreukaryotic cells e.g. yeast, unicellular eukaryotes such as Leishmainia,insect cells or mammalian cells or cell lines. Cells in liquid cultureor grown as colonies may be formed as E. coli, Bacillus subtilis,Streptococcus lactis, Streptococcus lividens, Lactococcus lactis,Staphylococcus aureas, Aspergillus niger, Picia pastoris, Saccharomycescerevisiae or Schizosaccaromyces pombe. All of the above are examples ofa sample comprising a target protein.

As mentioned above, key to the present invention is the finding that‘dirty’ samples can yield reliable information when undergoing thermalshift analysis. Thus, the sample at (a) is non-purified but there may becircumstances where a purified target protein has been added to a dirtystarting sample. The sample is not purified and contains components suchas other proteins, cell debris, nucleic acids etc., as described hereinin the context of “non purified target protein”.

Typically, a non-purified target protein has not been subjected to apurification process which would result in the purification of thetarget protein. Such a purification process may comprise of severalsteps and thus the non-purified target protein used in the presentinvention has not been subjected to all such necessary steps to producea purified protein. For example where the protein is present in atissue, steps of extraction, precipitation and separation e.g. bycentrifugation or chromatography may be used to purify the protein. Thenonpurified target protein of the present invention would not besubjected to all such steps and thus a purified target protein would notbe isolated. It is possible that the non-purified target protein couldhave been subjected to one or more steps e.g. the extraction step of apurification process, as long as the purification process was notcompleted and a purified protein was not isolated. The non-purifiedtarget protein is therefore typically present with other compounds orproteins and thus the target protein is not present in isolated form.

The term “test molecule” as used herein refers to any molecule orcompound, which is tested in the methods of the invention to determinewhether or not it is a ligand for the target protein. Alternativelyviewed, the test molecule is a potential ligand for the target protein.Thus, the test molecule or ligand may be a protein, polypeptide,peptide, RNA, or DNA molecule. In a particular embodiment, themolecule/ligand may be a drug or pharmaceutical product, a cellmetabolite or a hormone e.g. in serum. The test molecule or ligand maybe naturally occurring or may be synthetically or recombinantlyproduced, using any of the methods already described or discussedfurther below.

The test molecule used mayor may not bind to the target protein; in oneaspect the method of the invention determines or assesses whether aparticular molecule or compound is capable of binding to the targetprotein i.e. whether a test molecule or compound is a ligand. Thus, theinvention can be used to screen a small molecule library for moleculeswhich are capable of binding to the target protein. Some of themolecules tested may not bind, whereas others may bind to the targetprotein. Additionally, the method of the invention can be used toidentify variants of small molecules known to bind to the targetprotein, which can bind the target protein with higher affinity (oralternatively with lower affinity) where this is often reflected in thedegree of thermal stabilization. Thus, test molecules can be mutatedligands or known (or unknown) target protein binding partners. Theproduction of such mutated molecules is achieved by using any of themutation processes described herein.

Thus, in one aspect, the present invention provides a method foridentifying a ligand capable of binding to a target protein comprisingthe steps of:

(a) exposing a non-purified sample comprising said target protein and atest molecule to a series of different temperatures, including atemperature which is equal to or greater than the initial meltingtemperature of the target protein;

(b) processing the products of step a) in order to separate soluble frominsoluble protein and

(c) analysing either or both the soluble and insoluble protein fractionsof step b) for the presence of target protein, wherein said targetprotein is not detected on the basis of enzymatic activity of a tag,peptide, polypeptide or protein fused thereto.

The term “ligand” as used herein refers to a test molecule or moregenerally to a compound which is capable of binding to the targetprotein. A target protein may have a co-factor or physiologicalsubstrate bound thereto but methods of the invention investigate themelting point of a target protein bound to a ligand of interest ascompared to the target protein when not bound to that ligand (unboundtarget protein). The ligand of interest may bind elsewhere on theprotein or may compete for binding e.g. with a physiological ligand.Ligands of interest may be drugs or drug candidates or naturallyoccurring binding partners, physiological substrates etc. Thus, theligand can bind to the target protein to form a larger complex. Theligand can bind to the target protein with any affinity i.e. with highor low affinity. Generally, a ligand which binds to the target proteinwith high affinity may result in a more thermally stable target proteincompared to a ligand which binds to the target proteins with a loweraffinity. Typically, a ligand capable of binding to a target protein mayresult in the thermal stabilisation of that target protein by at least0.25 or 0.5° C. and preferably at least 1, 1.5 or 2° C.

Hence, when a test molecule is already known to bind the target protein(and thus is a ligand for the target protein), the method of theinvention can be used to assess the binding of the ligand to the targetprotein e.g. to determine the strength of the interaction. In thisaspect, the invention provides a method for assessing ligand binding toa target protein wherein said target protein is nonpurified comprisingthe steps of a) exposing a sample comprising said target protein andsaid ligand, to a temperature which is capable of causing or enhancingprecipitation of said target protein, b) processing the product of stepa) in order to separate soluble from insoluble protein and c) analysingthe soluble proteins of step b) for the presence of target proteinwherein said target protein is not detected on the basis of enzymaticactivity of a tag, peptide, polypeptide or protein fused thereto.

In order to assess or determine ligand binding to a non-purified targetprotein or to identify a ligand for a non-purified target protein, thetest molecule or ligand is typically added to the sample. However, it ispossible that the test molecule or ligand is already present in a samplecomprising the non-purified target protein e.g. is naturally occurring.Thus the invention may also provide a method for identifying a ligandwhich is capable of binding to a target protein wherein said targetprotein is non-purified comprising the steps of

(ai) adding a test molecule to said non-purified target protein

(a) exposing the product of step (ai) to a temperature which is capableof causing or enhancing precipitation of said target protein

(b) processing the product of step (a) in order to separate soluble frominsoluble proteins and

(c) analysing the soluble proteins of step (b) for the presence oftarget protein wherein the presence of the target protein indicates thatsaid molecule is bound to said target protein and is a ligand capable ofbinding to said target protein and wherein said target protein is notdetected on the basis of enzymatic activity of a tag, peptide,polypeptide or protein fused thereto.

Typically, where the test molecule (potential ligand) is presentextracellularly e.g. in solution, this may be simply added to thenon-purified target protein e.g. mixed together with the non-purifiedtarget protein where this is also in solution or dropped onto the targetprotein e.g. where the target protein is present in an aliquot ofharvested cells. Alternatively, the test molecule (potential ligand) maybe expressed recombinantly from a vector encoding the test molecule. Thestep of adding the test molecule may therefore involve transforming ortransfecting a cellular sample with the vector encoding the testmolecule and/or inducing expression of the test molecule from the vectorin a cellular sample once transformation or transfection has beencarried out. The step of adding the test molecule further includesinducing expression of a test molecule encoded by a gene naturallyoccurring in a cellular sample.

Further, where the target protein is present within a cell, the methodmay require an extracellular test molecule or ligand to be transportedinto the cell to contact the target protein. For test molecules orligands which bind to a target protein on the cell surface however,there is no need for transport into the cell. Alternatively, oradditionally, where the target protein is present in a cell (or on thecell surface), a step of lysis may be carried out before, simultaneouslyor after the test molecule or ligand has been added. Such a lysis stepallows contact between the target protein and the test molecule orligand and/or the later assessment of any binding between the testmolecule or ligand and target protein. Thus, any necessary lysis step isgenerally carried out before the separation step of the method of theinvention. It will be apparent that a step of lysis may only need to becarried out on samples where the target protein is comprised within acell. The lysis step may be thermal dependent i.e. the lysis may onlyoccur at a particular temperature e.g. at the end of a thermal cycle.

The lysis step of the present invention will have different requirementsdepending on whether the cells are subjected to heat treatment before orafter any lysis step. For cells subjected to lysis before heattreatment, preferably, the lysis step is non-denaturing, allowing targetproteins to retain a native i.e. correctly folded or native-likeconformation. This is referred to herein as native lysis. This can becarried out chemically or otherwise using reagents which are well knownin the art e.g. urea, lyzozyme containing buffers or detergents. Thedegree of lysis must be sufficient to allow proteins of the cell to passfreely out of the cell. Typically, when dealing with membrane boundproteins, lysis is performed in the presence of detergents oramphiphiles, for example Triton X-100 or dodecylmaltoside, to releasethe protein from the membrane. The lysis step can alternatively becarried out by freeze thawing the cells or colonies. More preferably,lysis is carried out using both native lysis buffer and freeze thawingthe cells. Preferably, the lysis buffer contains lysozyme, for examplesat 50-750 μg/ml, more preferably at 100-200 μg/ml. DNAse can also befound in native lysis buffer preferably at 250-750 μg/ml. Native lysisbuffer may contain for example 20 mM Tris, pH 8, 100 mM NaCl, lysozyme(200 lμg/ml) and DNAse I (750 μg/ml). For target proteins known to beinserted into cellular membranes, detergents would be added to the lysisbuffer at typical concentrations where they are known to solubilisemembrane-inserted proteins in a native form, such as 1%n-dodecyl-β-maltoside. Typically, the cells will be exposed to the lysisbuffer for 15-60 minutes, preferably around 30 minutes. The step offreeze thawing is preferably repeated, i.e. two or more cycles,preferably 3 or more cycles of freeze thawing are performed. In onepreferred embodiment lysis is achieved by a 30 minute incubation at roomtemperature with lysis buffer and three×10 minutes freeze thawing.

Typically, the percentage of cells lysed within a sample (e.g. a cellcolony or cell culture) during the lysis step is 5-100%. Thus, it is notnecessary when performing a step of lysis for all cells within a sampleto be lysed. Only a small percentage are required to be lysed in orderto release sufficient target protein to either contact with ligandand/or to be subjected to the separation step.

As discussed briefly above, it is possible that the test molecule orligand is already present in a sample comprising the target protein. Inthis instance it may be possible to investigate natural ligand bindingto a target protein e.g. by diluting the sample with buffer anddetecting any negative shift in thermal stability of the target proteinwhen a ligand is released.

The methods of the invention require that the non-purified sample isexposed to “a temperature which is capable of causing or enhancingprecipitation of said target protein”. This refers to a temperaturewhich is capable of causing or enhancing precipitation of target proteinin the absence of the test molecule (potential ligand) Likewise thenon-purified sample is exposed to “a temperature which is capable ofcausing or enhancing precipitation of the unbound target protein to agreater extent than it is capable of causing or enhancing precipitationof the target protein bound to said ligand”. “Unbound” refers to thetarget protein when not bound to, i.e. in the absence of, the ligand ofinterest.

Thus, as discussed previously, the inventors have found that proteins innon-purified form generally precipitate with a particular temperaturedependence, (i.e. having distinct melting temperatures) in a similarmanner to purified proteins, despite the varying conditions found withinnon-purified samples and particularly within cells. Therefore, theprotein may precipitate over a small temperature range. Occasionally,some proteins may undergo several transitions in their state duringheating over a temperature range indicating that there are several formsof the protein present in the sample (e.g. different spliced forms,phosphorylated forms, or bound to other proteins). In this situation, itis possible that a test molecule/ligand will not bind to all forms ofthe protein in all transition states. Hence, a test molecule or ligandmay only bind protein in one or more of its transition states. Thus, itis possible that a test molecule/ligand may only be able to thermallystabilise certain transition states or forms of the protein and thermalshifts in the stability will only be seen for these transition states.

Where a target protein precipitates over a small temperature range, theinitial melting temperature is the first temperature in the range andthe final melting temperature is the last temperature in the range.Thus, the initial melting temperature is the lowest temperature at whichtarget protein begins to precipitate e.g. at least 5% of the targetprotein is precipitated and the final melting temperature is the firsttemperature at which no soluble target protein is detected. e.g. lessthan 5% of target protein is in soluble form. Typically, at least 95% oftarget protein is melted and precipitated.

Therefore, when a target protein precipitates over a temperature range,the target protein may begin to precipitate or unfold at a particulartemperature at which point the amount of soluble target protein presentwill begin to decrease and the amount of insoluble target proteinpresent will increase (since thermal stability is linked to solubility).Therefore, some soluble protein may still be detectable at the initialmelting temperature until a slightly higher temperature is applied, atwhich point little or no soluble protein is detectable.

The final melting temperature for a protein is therefore a particulartemperature at which there is a significant decrease of soluble proteindetected, typically at least 95% of the protein is insoluble. Forproblematic proteins having multiple transitions, each of thesetransitions may result in a smaller amount of protein becominginsoluble, but this would still be significant enough to be measured(e.g. at least 10% of the protein becomes soluble at each transition).Where the protein precipitates over a small temperature range, where thepercentage of soluble protein decreases until no soluble protein isdetectable and thus the protein is completely unfolded or precipitated,an initial and final melting temperature can be determined. Hence, atthe initial melting temperature of such a temperature range i.e. thelowest temperature at which target protein begins to melt orprecipitate, at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,70, 75, 80, 85, 90 or 95% of the target protein may melt or precipitate.Alternatively viewed, at the initial melting temperature of atemperature range, the amount of soluble target protein detecteddecreases by at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,70, 75, 80, 85, 90 or 95%. Further, the amount of insoluble targetprotein present may increase by at least 5, 10, 15, 20, 25, 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or 95%.

It is also possible that a target protein may unfold and precipitate atone specific temperature. In this instance, preferably at least 95% ofthe target protein will be in insoluble form at a specific temperatureand hence the protein may not precipitate over a small temperaturerange. The initial melting temperature for such proteins may thereforebe close to the final melting temperature.

The temperature which can be applied in the present invention may be anytemperature from the initial melting temperature at which the targetprotein begins to unfold. Any temperature equal to or higher than theinitial melting temperature will be capable of causing or enhancingprecipitation of the target protein. Thus, a target protein with ahigher thermal stability due to ligand binding will generally not unfoldor precipitate at this temperature and a higher amount of solubleprotein will be detected as compared to target protein alone which haseither completely unfolded or begun to unfold. The temperature is thusdiscriminatory, causing or enhancing precipitation of the unbound targetprotein to a greater extent than it causes or enhances precipitation ofthe target protein bound to the ligand of interest.

The detection of an increased amount of soluble target protein at aparticular temperature when a test molecule is present as compared tothe amount of soluble target protein present when the test molecule isabsent is indicative that the molecule is a ligand for the targetprotein and that the test molecule is bound to the target protein. Wherethe temperature used in the present invention is the initial meltingtemperature or a temperature between the initial melting temperature andthe final melting temperature (i.e. not a temperature which results inat least 95% of the target protein being insoluble (the final meltingtemperature or a higher temperature than this)) it may be necessary tocarry out a control reaction simultaneously for target protein withoutligand present, in order to compare the amounts of soluble proteindetected in both cases, to detect the samples with ligand where anincreased amount of soluble target protein is present compared targetprotein alone. This is typically done by measuring the melting curve ofthe protein in similar non non-purified samples. However, where atemperature is used in the invention at which no or very little solubletarget protein is detected (i.e. target protein without ligand) e.g. thefinal melting temperature, there is no need to use a comparison orcontrol for every measurement. In this case, any detection of solubleprotein in the method indicates the presence of a thermally stable andhence ligand bound target protein. Such a temperature would typically beequal to or higher than the final melting temperature.

Additionally, the temperature can be chosen in the present invention toscreen for only ligands which bind to the target protein with a highaffinity. Thus, typically the higher the temperature at which solubleproteins and hence thermally stable ligand bound target proteins aredetected, the higher the affinity of the ligand binding to the targetprotein is likely to be. Hence, if only high affinity interactions arerequired to be detected, a temperature which is higher than the finalmelting temperature can be selected e.g. 3, 4, 5, 6, 7, 8, 9, 10 or more° C. higher than the final melting temperature. Thus, preferably, thetemperature selected would be higher than the final melting temperatureof a temperature range. Alternatively, if it is desired to identify allmolecules/ligands bound to the target protein, a lower temperature canbe used, for example one equal to the initial melting temperature of arange. Alternatively viewed, when selecting for high affinityinteractions, the discriminatory temperature of step (a) will be onewhich causes or enhances precipitation of unbound target protein to amuch greater extent than it causes or enhances precipitation of theligand bound target protein, e.g. at least 30% more, preferably at least50% more, more preferably at least 60, 70 or 80% more.

The binding affinity of the ligand to the target protein can bedetermined through performing the method steps described above at arange of varying ligand concentrations or target protein concentrations.In such methods, the sample treated in step (a) will have added theretoa known amount of target protein or ligand. One can plot a dose-responsecurve, and therefore determine the binding constant of the ligand (i.e.the concentration of ligand or target protein at which half of thetarget protein is bound to ligand). Such binding information obtained ina clinical, impure sample would provide a more accurate interpretationof the binding characteristics of the ligand to the target protein underphysiological conditions compared to information derived from puresamples. Such information could have useful applications to set dosingregimes for patients or to find a therapeutic window for a drug bystudies of apparent binding constants in different organs of the body.Thus, certain aspects of the invention may also comprise a further step:

d) repeating steps a) to c) with one or more (e.g. 2 or more, preferably3 or 4 or more) different concentrations of ligand or target protein.

The heating step can be carried out using any heat source which can heata sample to a particular temperature. Thus, where the non-purifiedtarget protein and test molecule (potential ligand) are in liquid form,then preferably the heating step may be carried out in a PCR machine.However, incubators, waterbaths etc may also be used. Where the targetprotein is in a cell colony, an incubator is preferably used to carryout the heating step.

The invention further encompasses applying a range of temperatures tothe target protein and test molecule and processing and analysing thetarget protein after incubation at each temperature in order to producea precipitation curve for each target protein and test moleculecombination. Thus, a target protein and ligand may be incubated at anytemperature range as long as one temperature is used which is capable ofcausing or enhancing precipitation of the target protein (i.e. withoutbound ligand). Preferably therefore, the temperature range appliedincludes incubating at the initial melting temperature or at atemperature higher than the initial melting temperature. By incubatingthe non-purified target protein and test molecule at a whole range oftemperatures, it is possible to determine the temperature at which thetarget protein precipitates when ligand is bound. Further, if a controlof non-purified sample without ligand is subjected to the sametemperature incubations, it is possible to identify ligand bound proteinsamples without prior knowledge of the target protein meltingtemperature. Preferably, any such heating of a control would be carriedout simultaneously to the heating of the non-purified sample and testmolecule/ligand. By using a precipitation curve, it is also possible todetermine ligands which have the greatest effect on thermal stabilitywhen more than one ligand is being investigated.

Typically a temperature range may be used to produce a precipitationcurve where the temperatures used are about 2, 3, 4, 5, 6, 7, 8, 9 or10° C. different from one another. Thus the target protein and testmolecule could be incubated at any one of more of 27, 30, 33, 36, 39,42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72 and 75° C. as long as one ofthe temperatures is equal to or higher than the initial meltingtemperature for the target protein. Where the target protein and testmolecule are heated over a temperature range, this can be carried out ina PCR machine where an initial temperature can be set and then increasedby the desired amount after a particular amount of time e.g. 1, 2, 3, 4or 5 minutes. As discussed previously, a small aliquot or amount ofsample (e.g. 1 or 2 μl) can be removed after heating at each temperaturein order that the solubility of the target protein can be analysed.Where the non-purified target protein is present in one or more cellcolonies, a portion of the colony may be lifted off after eachincubation e.g. by placing filter paper on the top of the colony.

In order to apply the method of the invention, it is necessary todetermine the melting temperature(s) of the target protein of interestwithout test molecule/ligand so that any thermal shift in the presenceof test molecule/ligand can be detected. Thus, the meltingtemperature(s) of the target protein can be determined before the methodof the invention is carried out or a simultaneous control reaction canbe carried out with the method of the invention where a range oftemperatures are applied to the control and to the target protein andmolecule e.g. as discussed above to produce a precipitation curve. TheTms (temperature at which 50% of protein is precipitated) of many targetproteins in purified samples are also known in the art and although theTms for non-purified target proteins are slightly different, these canoften be used as a guide for the melting temperatures of non-purifiedproteins.

“A temperature capable of causing or enhancing precipitation” of targetprotein therefore refers to a temperature or a temperature range asdiscussed above at which there is an increase in the precipitation oralternatively viewed the unfolding or melting of a target protein ascompared to target protein at a lower temperature. The temperature isgenerally an increased temperature compared to the temperature at whichthe target protein is usually found e.g. 37° C. for target proteinswithin a patient. Thus the temperature applied is typically above 37°C., preferably above 40° C., e.g. above 50° C.

The temperature used in the invention thus preferably causes an increasein precipitation of the target protein by at least 5, 10, 15, 20, 30,40, 50, 60, 70, 80, 90 or 95%. An increase in protein precipitationusually results in insoluble protein being produced and thusalternatively viewed, the temperature used in the invention may cause anincrease in the amount of insoluble target protein present e.g. anincrease of at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90or 95%. In the present invention, any enhancement of precipitation maybe measured by measuring a decrease or reduction in the amount ofsoluble target protein present e.g. a reduction of at least 5, 10, 15,20, 25, 30, 35, 40, 50, 60, 70, 80, 90 or 95%. Measurement of thisdecrease could for example be done using dot-blots or ELISA-experimentwhere the amount of bound antibody can be quantified using e.g.integration of fluorescence signals of florescence labelled antibodies.

The method of the invention further requires the use of a separationstep (b) to separate soluble from insoluble proteins. The separationstep can involve any separation method which is capable of separatingsoluble from insoluble protein. For example, a step of centrifugationcan be used as described above or in a preferred embodiment, a step offiltration may be used. Thus, a filter can be used to separate solublefrom insoluble proteins where soluble proteins will pass through afilter. Standard filter membranes can be used for filtering heatedsamples where the filters will typically have a pore size from 0.015 μmto 12 μm, preferably from 0.35 to 1.2 μm, more preferably from 0.45 μmto 0.8 μm. Preferably the filters have pore sizes below 4.0 μm,typically below 2.0 μm, more preferably below 1.0 μm. When the targetprotein is produced or expressed in cells, such as bacteria e.g. E.coli, an optimal pore size may be 0.1-1.5 μm. Where is target protein isfrom a eukaryotic cell or sample, preferred pore sizes may be larger. Itwill be appreciated that filters are manufactured and marketed as havinga particular pore size but the manufacturing process may occasionallyresult in a few smaller or larger pores; the sizes listed, which referto the diameter, are thus the most common pore size of a given filter.Although reference is made to a range of potential pore sizes, anysingle filter will usually have one designated pore size e.g. 0.45 μm.Suitable filters are Super and GH polypro (from Pall) and Nucleopore(From Whatman).

It will be appreciated that target proteins from eukaryotic andprokaryotic samples and from different cell types may require the use offilters with different pore sizes. Selection of a suitable filter iswell within the competency of someone skilled in this field. Forexample, it is possible to select an appropriate pore size, by using aset of test proteins for the desired cell type or sample andinvestigating their behaviour with filters of varying pore sizes.

As discussed previously, where the target protein is present within acell, a step of cell lysis may be carried out prior to the separationstep. Cell lysis will also be required when the sample is a cell sampleand target protein is added thereto in order to assay for the presenceof a ligand. When the present method is carried out on cell colonies,the lysis may be carried out directly on those colonies i.e. there is noneed to pick the colonies and grow them in liquid culture (although thiscan be done). In this instance, it is preferred that the separation stepis one of filtration. Further, where the method is performed on cellcolonies, preferably, the filter paper is overlayed on the colonies tolift the colonies from the semi-solid or solid growth media.Alternatively, filters could be placed on the growth media and cellsseeded directly onto the filter, the filter could then simply be liftedoff with the colonies already on it. Preferably, the lifting of thecolonies in this way can be carried out prior to the lysis step. Asindicated above, the lysis can be carried out directly on the colonieson a filter. The filter with colonies attached can be treated with lysisbuffer or overlaid on other membranes/filters treated with lysis buffer.

Filtration can also be carried out for liquid cultures of cells e.g.liquid cultures growing in a multi well plate e.g. a 96 well plate.

Filtration is carried out after any necessary lysis step is performed.It will be appreciated however that filtration and lysis may occursimultaneously when considering a whole colony since some cells mayundergo lysis before others and hence may be filtered before or at thesame time as others are lysed.

Preferably, where the separation step is filtration, proteins which passthrough the filter are held on a solid support, e.g. a capture membrane,to allow screening/detection of the target protein(s) and then to allowthe identification of sample(s) containing the target protein bound toligand. Such capture membranes may typically comprise nitrocellulose.However, it will be appreciated that it is the first filter thatseparates soluble from insoluble protein in this method. In a preferredembodiment, proteins can simply be allowed to pass through the filter,possibly as a result of capillary action. In another embodiment, forcemay be applied vertically on the filter paper, wherein such forces caninclude the application of pressure or vacuum.

The capture membrane can fix the soluble proteins from the individualsample(s) and in this way, it is possible to multiplex this method.Thus, the positions of the target protein(s) on the capture membrane canbe compared to the filter which either carries the original cellcolonies, if the method is being carried out on cell colonies, or thesample spots. Thus, from the filtration blot, it is possible to trackback and identify the original samples comprising the target protein andbound ligand. To aid in the process of identifying colonies comprisingtarget protein bound to ligand, positive controls can be used. These areclearly seen on the final colony filtration blots and can enable themembrane/blot to be correctly orientated with the original colonies.Hence, after any filtration step is carried out, a solid support such asa capture membrane allows the ready identification of samples havingtarget protein bound to ligand.

In another embodiment, the filter with heat treated sample(s) can beplaced sample side down and a (nitrocellulose) capture membrane can thenbe placed on top of the filter and several layers of filter paper (andpaper towels) can be placed on top of this. Force can then be applied tothe top of this “sandwich” and ideally transfer buffer poured around thebottom to facilitate filtration and transfer of proteins onto thecapture membrane.

In another embodiment, the filter is placed sample side up onto acapture membrane and a vacuum is applied to “pull” protein through thefilter paper and onto the capture membrane.

Alternatively to filtration and centrifugation, affinity capture ofsoluble protein can be carried out. Many antibodies and affinityreagents that recognise the folded structure of the protein will bindthe soluble protein with much higher affinity than the unfolded andprecipitated protein. Also the recognition of smaller tags such aspoly-Histidine tags binding to metal conjugates will often correlatewith solubility when these tags are less accessible in the precipitatedprotein. Antibodies, metal conjugates and other affinity reagents can belinked to magnetic beads or column resin which is mixed with the heattreated non-purified sample. This mix can in a subsequent step be put inan appropriate valve and washed to remove insoluble protein when thisdoes not have high affinity to the affinity reagent. The amount ofprotein bound to the affinity reagent, can subsequently be measuredusing for example Bradford techniques, gel electrophoresis, Elisa orsurface plasmon resonance detection.

According to the methods of the invention it is possible to analyseeither (or both) the insoluble or soluble fractions for the presence oftarget protein. The insoluble fraction is preferably solubilised priorto analysis, for example, as described in Example 3, the precipitatedproteins may be dissolved in loading buffer prior to application to theseparation gels. Preferably the methods of the invention involve a step(c) of analysing the soluble proteins for the presence of targetprotein. Thus, the soluble proteins obtained after the step ofseparation are preferably analysed for the presence of target protein.Hence, if a centrifugation separation step was carried out, thesupernatant can be analysed for the presence of target protein and wherea filtration separation step was carried out, the proteins which passthrough the filter i.e. the filtrate can be analysed for the presence oftarget protein.

The target protein can be detected by various different methods. Thus,target proteins can be detected using various tags which are well knownin the art, e.g. histidine tag, VS tag, T7 tag, FLAG tag or any shortprotein sequence to which a specific antibody is available, thioredoxinand maltose binding protein. Tags are preferably between 1-100 aminoacids in length, preferably between 1-70, 2-50, 1-30 or 1-20 amino acidsin length. More preferably, tags can be 3, 4, 5, 6, 7, 8, 9 or 10 aminoacids in length. However, the target protein is not detected on thebasis of any enzymatic activity of a tag, peptide, polypeptide orprotein which is fused to the target protein. Thus, target proteins arenot detected using an enzymatic activity exhibited by any such tags orproteins fused to the target protein, e.g. where the enzymatic activityresults in the production of a detectable signal. For example, fusiontags that possess enzymatic activity such as green fluorescent protein,horseradish peroxidase, luciferase and glutathione-S-transferase are notused in the present invention to detect the target protein. Thus,although it is possible for any tag/protein to be fused to the targetprotein, the target protein is not detected using the enzymatic activitypossessed by any such tags or proteins. Thus, in the case of a GFP tag,fluorescent green light is produced by an enzymatic reaction and henceit is specifically excluded in the present invention, for a targetprotein to be detected using such a reaction. Hence, the detection ofthe target protein by fluorescence produced from a GFP tag is excluded,since such fluorescence is the result of enzymatic activity possessed bythe tag. Further, in a preferred embodiment, the target protein of theinvention is not fused to a reporter protein with enzymatic activity. Ina particularly preferred embodiment, the target protein is not fused toGFP. Thus, alternatively viewed, it is preferred that any tag fused tothe target protein is a non-protein tag.

For a tag to be fused to the target protein, it is generally transcribedand translated with the target protein as a single molecule. Thus,antibodies which bind to the target protein and which may be labelledwith HRP etc are not considered to be fused to the target protein. Insuch cases, the target protein may be detected using the HRP tag sincethis is not part of a fusion molecule with the target protein.

Thus, tags can be attached to a target protein by expressing suchproteins as fusion proteins. As such, short tags are preferred, to allowproteins of interest to maintain a native-like conformation. Further,C-terminal tags are preferred, although N-terminal His tags are alsoused. It will be appreciated that a detection step involving the use ofa tag fused to a target protein can only be used where the targetprotein is derived from a recombinant expression system. Therefore,generally this detection method will not be used when the target proteinis for example obtained from a patient.

Target proteins can further be detected via fusion tags which act as thesubstrate in enzymatic detection methods, His tags being particularlysuitable in this regard. For example, INDIA His Probe-HRP (Pierre,Rockford Ill., USA) can be used for detection wherein the target proteinis either poly-histidine tagged or is histidine rich and where thetarget protein is detected by Nickel activated derivative of horseradishperoxidase which binds to His tags. Target proteins may also be detectedon the basis of their own enzymatic activity.

Detection may alternatively be based on affinity binding between thetarget protein and a detection moiety or between a tag fused to thetarget protein and a detection moiety, for example an antibody, antibodyfragment or affibody (non Ab based protein binding partner). Preferably,target proteins may be detected using antibodies, monoclonal orpolyclonal, either directed to a tag or directly to the target protein(expressed on its own or as a fusion). Antibodies which are directed tothe target protein are typically used to detect a target protein from apatient sample. Such methods allow for rapid and reliable analysis of awide variety of target proteins, including those which themselvespossess no catalytic activity. Target protein can also be detected usingsemi quantitative mass spectrometry (MS). In a fourier transform ioncyclotron resonance experiment using an orbitrap instrument typically1000-2000 proteins can be detected simultaneously in a sample from alysate. In a preferred embodiment a temperature scan of cells followedby lysis, filtration and in a final step the detection of all remainingsoluble protein using mass spectrometry, at each temperature of thescan, allows precipitation curves to be measured in parallel for manyproteins. This global proteome melting curve analysis could for examplebe used to detect so called off target effects of drugs, i.e. to monitorwhich other proteins in the cell appear to bind the drug. This globalproteome melting curve analysis could also be used when searching fordrug targets for drugs or drug candidates for which the drug target isunknown. For example, compound library screening direct on cells canidentify compounds that generate preferred phenotypes in these cellsindicative that the compound effects processes in the cell that areuseful as drug targets for a certain disease. However, it is normallyvery challenging to identify with which protein or proteins in the cellthe drug candidate interact. The global proteome melting curve analysisfor thermal shift changes allow this to be performed for the proteinswhich are available at sufficient level to be detectable with MS.

Molecule/ligand binding to target proteins can be investigated in arecombinant expression system. Thus, genes/cDNAs/coding regions for thetarget protein can be transformed or transfected into expression systemsin vectors/constructs, such as plasmids, viral vectors, cosmids andYACS. Such vectors may contain regulatory sequences and other elementswell known in the art. For example, the gene/cDNA/coding region may beplaced under the control of a promoter in a vector. Promoters used aregenerally capable of expressing the target protein within a particularhost. In a specific embodiment, the promoter used is inducible i.e. theexpression of the target protein can be controlled. Such induciblepromoters/systems include lac wherein induction of expression iscontrolled by the addition of IPTG and tet on/off, wherein the inductionof expression is controlled by the presence/absence of tetracycline andothers are known in the field.

As described previously, the method of the invention may be used toscreen libraries of small molecules for those which will bind to atarget protein. In a preferred embodiment, this is carried out usingmulti-well plates where each compound of the library is added to analiquot of cells, or a cell lysate. Alternatively libraries of mutanttarget protein may be screened to determine a mutant target proteinwhich shows altered binding to a particular ligand. For example, mutanttarget proteins can be identified which have a closer or tighterassociation with a ligand than wildtype target protein. Where mutanttarget proteins are being assessed, measurements of the stability of theprotein without ligand are desirable to decide whether the stabilisationis due to the ligand interaction or due to the mutant itself being morestable i.e. the mutation having a stabilising effect on the mutantprotein. If the ligand is another protein, the stability measurementcould instead be carried out on this non-mutant protein, where mutatedprotein variant can be selected which stabilises the non-mutatedprotein. This could, for example, be used to mature binding proteins(i.e. the ligands) such as, for example, antibodies, FAB-fragments,single chain antibodies or affibodies where random mutations are addedto the binding protein and variants with apparent improved binding aredetected by measuring improved stabilization of the non-mutated protein.In such a way, higher affinity binders could be selected from loweraffinity binders. When binding proteins can serve as protein drugstargeted against e.g. specific receptors or cytokines, the method couldbe used to improve the affinity of such binders to the drug target ofthe protein drug.

Many different methods of mutagenesis are known in the art which couldbe employed to create a variant of the target protein or a library ofvariants of target protein. Possible procedures include truncation ofthe sequence, use of an exonuclease enzyme, introduction of a randomizedsite mutations using e.g. error prone PCR, introduction of randomisedcassette or site-directed mutagenesis. For truncations, the number ofnucleotides removed may be less than 2000, preferably less than 1000 andmore preferably less than 800. Introduction of a randomised cassette formutagenesis preferably uses a cassette containing less than 100nucleotides.

Mutagenesis may be carried out on several copies of a nucleic acidsequence encoding the target protein so that a set of different mutatedsequences can be screened, hence increasing the probability ofidentifying a target protein variant with the desired ligand bindingproperties. The use of random mutagenesis is especially preferred wherethere is no prior knowledge of which particular mutations may yield avariant which for example binds to the ligand more tightly i.e. has ahigher affinity for the ligand.

Libraries of proteins can be created where the coding region has beenrandomly mutagenised and where different length constructs have beengenerated by erase-a-base or random priming reactions.

Thus, the methods of the present invention can be used to detect targetprotein variants which have altered and preferably have increased orhigher affinity binding to a ligand. Additionally, the methods of theinvention can be used to determine whether a target protein in a cellculture or patient sample will interact with a particular test moleculee.g. a drug. Hence, a preferred use of the method is to determinedrug-protein interactions in cell culture during the drug developmentcycle to confirm that the drug binds to the target protein in this celltype. Similarly the method can be used to monitor drug binding tonon-desired proteins, so called off-target binding. Another preferreduse of the method is to determine drug-protein interactions in patientsamples (e.g. tissue, blood, lymph etc.), to provide an indication as towhether a particular drug therapy will be effective for that patient. Ifa tissue sample is to be examined, then the method of the invention mayalso incorporate a step of extracting a target protein from the tissue.Additionally or alternatively, a step of lysis may be used. Appropriatelysis conditions are described above.

Once a target protein and ligand interaction has been detected in anonpurified sample using the method of the invention, it may bedesirable to identify the sequence or structure of the target protein,particularly if target protein variants have been investigated.Alternatively, as discussed above, the results obtained may be used todetermine whether a drug therapy is likely to be effective in a patientand thus to tailor the therapy provided to a patient.

The binding of a high affinity drug to an established drug target, asshown in e.g. Examples 4, 5, 6 and 7, typically leads to a stabilisationof the target protein as supported by the positive shift of the meltingtemperature to a higher temperature. However, there are also ligandsthat, upon binding to the target protein, cause a negative shift of themelting temperature to a lower temperature, i.e. destabilisation. Forexample, negative shifts can be seen for ligands which formcovalent-type bonds (including some metals) to a target protein. It ispresumed that the binding energy of a covalent bond, and theenergetically unfavourable strains generated by forming such a bond,could, in some cases, promote the destabilisation of a protein. Forexample, Ericsson et al. (Anal Biochem 357 (2006) pp 289-298) show thatcompounds which contain heavy metal atoms, such as lutetium (III)chloride hexahydrate, are able to destabilise a number of bacterialproteins upon binding.

Thus, in a further aspect, the invention provides a method ofdetermining whether a non-purified sample contains a target proteinbound to a ligand of interest, wherein said ligand is not a fusionprotein, comprising the steps of:

a) exposing said non-purified sample to a temperature which is capableof causing or enhancing precipitation of the target protein bound tosaid ligand to a greater extent than it is capable of causing orenhancing precipitation of the unbound target protein;b) processing the product of step a) in order to separate soluble frominsoluble protein; andc) analysing either or both the soluble and insoluble protein fractionsof step b) for the presence of target protein, wherein said targetprotein is not detected on the basis of enzymatic activity of a tag,peptide, polypeptide or protein fused thereto.

Another further aspect of the invention provides a method of determiningwhether a non-purified sample contains a target protein bound to aligand of interest comprising the steps of:

a) exposing said non-purified sample to a temperature which is capableof causing or enhancing precipitation of the target protein bound tosaid ligand to a greater extent than it is capable of causing orenhancing precipitation of the unbound target protein;b) processing the product of step a) in order to separate soluble frominsoluble protein; andc) analysing the soluble protein fraction of step b) for the presence oftarget protein, wherein said target protein is not detected on the basisof enzymatic activity of a tag, peptide, polypeptide or protein fusedthereto.

In the above aspects, one would expose the non-purified sample to atemperature capable of causing or enhancing precipitation of the targetprotein bound to ligand, because the target protein bound to thedestabilising ligand would precipitate at a lower temperature comparedto the unbound target protein. Therefore, at the distinguishingtemperature described in step a), one would expect to find more of thebound protein in the insoluble protein fraction, and more of the unboundprotein in the soluble protein fraction.

Discussions of the various features of the methods of the invention andpreferred embodiments set out in relation to stabilisation caused byligand binding apply, mutatis mutandis, to these aspects of theinvention where ligand binding causes destabilisation.

In some instances, there might be a physiological substrate orco-factor, such as ATP or NADP, present in a cell lysate, which binds tothe target protein even before the ligand is added to the sample. When aligand of interest is added to such a lysate, the shift of the meltingcurve towards higher temperatures will typically be smaller, as comparedto the case when no physiological ligand is present in the lysate. In anextreme case, a very low affinity ligand (typically giving smallpositive thermal shifts) such as an early drug lead candidate, could atvery high concentrations compete out a stronger physiological ligand(typically giving large positive thermal shifts) such as NADP. Thereplacement of the physiological ligand could, in such a case, lead to anegative shift, i.e. a shift to a lower melting temperature, when theapparent shift is the difference between the shifts of the two ligandbound forms of the protein. Under such circumstances, because thenegative shift would be detectable as a decrease in the meltingtemperature of the target protein, the aspects of the invention relatingto the ligand causing destabilisation would apply here.

The invention further encompasses an instrument for use in the methodsof the invention wherein said instrument comprises a heating means, ameans for separating soluble from insoluble protein and a means foranalysing protein for the presence of target protein, e.g. for analysingsuitable protein.

Alternatively viewed, an instrument adapted in use to carry out themethod of the invention comprising a heating means, a means forseparating soluble from insoluble protein and a means for analysing(e.g. soluble) protein for the presence of target protein, isencompassed.

Further, the invention is directed to the use of an instrumentcomprising a heating means, a means for separating soluble frominsoluble protein and a means for analysing (e.g. soluble) protein forthe presence of target protein in the methods of the invention.

The instruments are arranged such that a sample is first contacted withthe heating means, then separation means and finally analysing means.

The term “a heating means” as used herein refers to any heat sourcewhich is capable of heating a sample to a particular temperature. Thus,the heating means may consist or comprise of a hot plate which can beprogrammed to heat a sample to a particular temperature, e.g. a PCRmachine can be used to heat a sample in this way. Further, a heatingmeans could comprise an incubator or a water bath.

The term “a means for separating soluble from insoluble protein” refersto any known apparatus which is capable of separating soluble andinsoluble protein. Thus the means may comprise a filter paper wheresoluble protein will pass through the filter paper. Alternatively, themeans may comprise an apparatus which is capable of imparting acentrifugal force on the heated sample e.g. a centrifuge. Additionally,the means may comprise an apparatus which is capable of affinity captureof the soluble protein. Such an apparatus may comprise antibodies orother affinity reagents which are capable of recognising the foldedstructure of the soluble protein. Antibodies, metal conjugates or otheraffinity reagents may be linked to magnetic beads or column resin.Insoluble protein can be removed by washing.

The term “means for analysing (e.g. soluble) protein for the presence oftarget protein” as used herein refers to any apparatus which would becapable of detecting the target protein. Thus this could refer to a massspectrometer but more preferably may refer to the apparatus required toe.g. detect an antibody labelled with HRP or a fluorescent moleculebound to the target protein (i.e. nitrocellulose membrane or afluorimeter). Further, the means for analysing protein for the presenceof target protein may comprise an affinity column for binding targetprotein. The means for analysing protein for the presence of targetprotein may further comprise any of the reagents necessary to detect thetarget protein, or alternatively, these may be provided separately.

Finally, the present invention encompasses the use of a kit in themethods of the invention which comprises an antibody and/or anon-protein tag.

The invention will now be further described in the followingnon-limiting Examples in which:

FIG. 1 shows the assessment of the presence of soluble protein for threedifferent proteins expressed in an E. coli sample after exposure to arange of different temperatures. The known melting temperatures of thepurified proteins are shown on the right hand side of the figure.

FIG. 2 shows the assessment of the presence of soluble PIK3C3-proteinafter the addition of the ligands Wortmannin and3-[4-(4-Morpholinyl)thieno[3,2-d]pyrimidin-2-yl]-phenol (Compound 15e)(+). Reference sample without added ligand are also shown (−). A thermalshift can be seen in the samples with ligands The lanes with proteinsplus ligand are more thermally stable than proteins without ligand.

FIGS. 3a and 3b shows the Western blot membranes of targets cyclindependent kinase-2 (CDK-2) (FIG. 3a ) and protein kinase C (PKC) (FIG.3b ). The dark bands indicate that the presence of soluble protein wasdetected up to a specific temperature and become fainter and ultimatelydisappear as the temperature is increased (from left to right). Thepellet containing precipitated protein from the highest temperature wasdissolved in loading buffer and loaded in the last lane of the gel inorder to show the presence of the target protein in this fraction.

FIG. 4 shows the levels of soluble thymidylate synthase (TS),dihydrofolate reductase (DHFR), CDK-2 or PKC protein present afterexposure to a range of different temperatures in mammalian cellextracts. The X axis represents the exposed temperature (° C.) and the Yaxis represents the integrated intensity from the Western blots.

FIG. 5 shows the thermal melting curve from human cell extracts ofsoluble DHFR protein after the addition of the inhibitor methotrexate(♦). Reference sample without inhibitor is also shown (▪). The X axisrepresents the exposed temperature (° C.) and the Y axis represents theintegrated intensity from the Western blots.

FIG. 6 shows the thermal melting curve from human cell extracts ofsoluble T8 protein after the addition of the inhibitor raltitrexed (+).Reference sample without inhibitor is also shown (●). The X axisrepresents the exposed temperature (° C.) and the Y axis represents theintegrated intensity from the Western blots.

FIGS. 7a and 7b shows the thermal melting curve of soluble methionineaminopeptidase-2 after the addition of the ligand TNP-470 (x) eitherfrom cow liver extract (FIG. 7a ) or from human cell extract (FIG. 7b ).Reference sample without ligand is also shown (O). The X axis representsthe exposed temperature (° C.) and the Y axis represents the integratedintensity from the Western blots.

FIG. 8 shows the dose response curve of TNP-470 treatment of cow liverextract. The X axis represents the concentration of TNP-470 added andthe Y axis represents the integrated intensity from the Western blots.

FIG. 9 shows the dose response curve of TNP-470 created by spiking withcell lysate containing target protein. The X axis represents theconcentration of TNP-470 added and the Y axis represents the integratedintensity from the Western blots.

FIG. 10 shows the thermal melting curve of either soluble V600E variantB-raf protein (♦) or wild-type B-raf protein (▴) after the addition ofthe ligand SB590885. Reference samples without ligand are also shown,both for the V600E varient B-raf protein (▪) and the wild-type B-rafprotein (x). The X axis represents the exposed temperature (° C.) andthe Y axis represents the integrated intensity from the Western blots.

Example 1 Determination of Melting Temperatures of Four Test Proteins

Three human soluble protein expression constructs in an expressionvector with an N-terminal His-tag were used in order to determine themelting temperature of each protein in the cell. This was done byexposing the protein-containing cell to a panel of increasingtemperatures and after each temperature step spotting the cells onto a“lysis/filtration sandwich” soaked in lysis buffer. By using thislysis/filtration step, the soluble protein (up to the protein's specificmelting temperature) could be detected on a capturing nitrocellulosemembrane as dark spots, whereas precipitated protein (i.e., above itsspecific melting temperature) was not able to pass through the filtermembrane and could therefore not be detected.

Materials and Methods

Liquid cultures of E. coli cells overexpressing the three proteins ofinterest were started by inoculating 1 ml Luria-Bertani broth (LB)(Formedium Ltd., UK) containing 50 μg/ml kanamycin (Sigma-Aldrich Co.,USA) and 35 μg/ml chloramphenicol (Duchefa Biochemie, The Netherlands)with frozen E. coli from glycerol stocks in a 96-well deep-well plate(Porvair Plc., UK). The cultures were incubated on a shaking boardovernight at 700 rpm and +37° C. The following day 100 μl of eachovernight culture was transferred to a corresponding well of a new96-well deep-well plate containing 900 μl LB, 50 μg/ml kanamycin, and 35μg/ml chloramphenicol. The cultures were incubated on a shaking board at700 rpm and +37° C. After 1.5 hours the temperature was lowered to +18°C. (30 min.), and protein expression was induced by adding 100 mM IPTG(Anatrace/Affymetrix Co., USA). The cells were grown overnight on ashaking board at 700 rpm and +18° C. The cells were pelleted bycentrifugation the following day at 1500 g for 2 min. and 900 μlsupernatant was removed from each well by aspiration and discarded. Thecell pellets were resuspended in the remaining 100 μl of medium (i.e.,concentrated 10-fold). The cell suspensions were transferred to 8-tubePCR strips (Applied Biosystems, UK) and placed in a thermocycler. Thefollowing temperature program was used: +27° C.-+75° C. with 3° C.increments and a 3 min. hold at each step. After the 3 min. hold at eachtemperature the thermocycler was paused, and 2 μl of each cellsuspension was quickly spotted onto a “lysis/filtration sandwich”consisting of Durapore filter membrane with 0.45 μm pore size (MilliporeInc., USA) (top layer), Protran BA 45 nitrocellulose membrane(Schleicher & Schuell, Germany) (middle layer), and 3 MM Whatman paper(VWR Int'l. Ltd., UK). The “lysis/filtration sandwich” was soaked innative lysis buffer (20 mM Tris-HCl pH 8.0, 100 mM NaCl, 10 mg/mlLysozyme (Sigma-Aldrich Co., USA), 25 U/μl Benzonase nuclease (Novagen,Denmark) and Complete protease inhibitor EDTA-free tablet (Roche,Switzerland). After spotting the cells onto the “lysis/filtrationsandwich”, the abovementioned procedure was repeated at each temperaturestep. After spotting the last cell aliquot the “lysis/filtrationsandwich” was incubated for 15 min. at room temperature in order toallow complete lysis and liquid cellular material transfer through thefilter membrane. The “lysis/filtration sandwich” was thereafter frozenat −80° C. for 10 min. and then thawed for 10 min. at +37° C. Thisfreeze/thaw procedure was repeated 3 times. The nitrocellulose membranewas blocked in TBST buffer (20 mM Tris-HCl pH 7.5, 500 mM NaCl, 0.05%Tween-20) containing 1% BSA (VWR Int'l. Ltd., UK) for 1 hour. The blotwas then washed 3 times for 10 min. in TBST with some agitation(tabletop shaker). The membrane was incubated for 1 hour with INDIAHisProbe-HRP (Thermo Scientific, USA) diluted 1:5000 in TBST. The blotwas then washed 3 times for 10 min. in TBST. Chemiluminescent detectionof target protein expression level in each spot on the blot wasperformed using SuperSignal West Dura (Pierce) Extended DurationSubstrate (Thermo Scientific, USA). Chemiluminescence was detected andrecorded using a CCD camera (BioRad Laboratories, Inc., USA).

Results

The dark spots indicating the presence of soluble protein were detectedup to a specific temperature; they became fainter and ultimatelydisappeared at higher temperatures (FIG. 1). A comparison with previousdata on the melting temperatures of the three IMAC-purified proteins(right panel) showed good correlation between the two sets of results.The melting points are not expected to be exact as the proteins havedifferent solvent environment in a cell and in a purification buffer.This experiment shows that these proteins have distinct melting pointsin the cellular environment and that these melting points can be easilydetected by monitoring the precipitation of the protein.

Example 2 Detection of an Increase of Melting Temperature after Bindingto a Ligand

An expression construct of human soluble PIK3C3 protein in an expressionvector with N-terminal His-tag was used to investigate a possibleincrease in melting temperature of the PIK3C3 construct after additionand binding of either of two PIK3C3 specific inhibitors; Wortmannin andCompound 15e. After treatment with or without one of the two inhibitorsthe cells expressing the PIK3C3 constructs were exposed to a panel ofincreasing temperatures and after each temperature step the cellsexpressing the proteins were spotted onto a “lysis/filtration sandwich”soaked in lysis buffer. By using this lysis/filtration step on the“lysis/filtration sandwich” the soluble protein (up to the construct'sspecific melting temperature) could be detected on a capturingnitrocellulose membrane as dark spots, whereas precipitated protein(i.e., above its specific melting temperature) was not able to passthrough the filter membrane and could therefore not be detected. Meltingtemperatures of constructs treated with Compound 15e or Wortmannin werecompared with those of untreated samples.

Materials and Methods

Liquid cultures of E. coli cells overexpressing PIK3C3 constructs werestarted by inoculating 1 ml Luria-Bertani broth (LB) (Formedium Ltd.,UK) containing 50 μg/ml kanamycin (Sigma-Aldrich Co., USA) and 35 μg/mlchloramphenicol (Duchefa Biochemie, The Netherlands) with frozen E. colifrom glycerol stocks in a 96-well deep-well plate (Porvair Plc., UK).The cultures were incubated on a shaking board overnight at 700 rpm and+37° C. The following day 100 μl of each overnight culture wastransferred to a corresponding well of a new 96-well deep-well platecontaining 900 μl LB, 50 μg/ml kanamycin, and 35 μg/ml chloramphenicol.The cultures were incubated on a shaking board at 700 rpm and +37° C.After 1.5 hours the temperature was lowered to +18° C. (30 min.), andprotein expression was induced by adding 100 mM IPTG(Anatrace/Affymetrix Co., USA). The cells were grown overnight on ashaking board at 700 rpm and +18° C. The cells were pelleted bycentrifugation the following day at 1500 g for 2 min. and 900 μlsupernatant was removed from each well by aspiration and discarded. Thecell pellets were resuspended in the remaining 100 μl of medium (i.e.,concentrated 10-fold). For each of the experiments 1 mM of the PIK3C3inhibitor Compound 15e (Santa Cruz Biotechnology, Inc., USA) or 500 μMWortmannin (Santa Cruz Biotechnology, Inc., USA) dissolved in DMSO(Sigma-Aldrich Co., USA) or the equivalent volume (1 μl and 0.5 μlrespectively) of pure DMSO was added and the samples were gentlyagitated for 30 min. at room temperature. The cell suspensions weretransferred to 58-tube PCR strips (Applied Biosystems, UK) and placed ina thermocycler. The following temperature program was used: +27° C.-+75°C. with 3° C. increments and a 3 min. hold at each step. After the 3min. hold at each temperature the thermocycler was paused, and 2 μl ofeach cell suspension was quickly spotted onto a “lysis/filtrationsandwich” consisting of Durapore filter membrane with 0.45 μm pore size(Millipore Inc., USA) (top layer), Protran BA 45 nitrocellulose membrane(Schleicher & Schuell, Germany) (middle layer), and 3 MM Whatman paper(VWR Intl Ltd., UK). The “lysis/filtration sandwich” was soaked innative lysis buffer (20 mM Tris-HCl pH 8.0, 100 mM NaCl, 10 mg/mlLysozyme (SigmaAldrich Co., USA), 25 U/μl Benzonase nuclease (Novagen,Denmark) and Complete protease inhibitor EDTA-free tablet (Roche,Switzerland). After spotting the cells onto the “lysis/filtrationsandwich”, the abovementioned procedure was repeated at each temperaturestep. After spotting the last cell aliquot the “lysis/filtrationsandwich” was incubated for 15 min. at room temperature in order toallow complete lysis and liquid cellular material transfer through thefilter membrane. The “lysis/filtration sandwich” was thereafter frozenat −80° C. for 10 min. and then thawed for 10 min. at +37° C. Thisfreeze/thaw procedure was repeated 3 times. The nitrocellulose membranewas blocked in TBST buffer (20 mM Tris-HCl pH 7.5, 500 mM NaCl, 0.05%Tween-20) containing 1% BSA (VWR Int'l. Ltd., UK) for 1 hour. The blotwas then washed 3 times for 10 min. in TBST with some agitation(tabletop shaker). The membrane was incubated for 1 hour with INDIAHisProbe-HRP (Thermo Scientific, USA) diluted 1:5000 in TBST. The blotwas then washed 3 times for 10 min. in TBST. Chemiluminescent detectionof target protein expression level in each spot on the blot wasperformed using SuperSignal West Dura (Pierce) Extended DurationSubstrate (Thermo Scientific, USA). Chemiluminescence was detected andrecorded using a CCD camera (BioRad Laboratories, Inc., USA).

Results

The dark spots indicating the presence of soluble protein were detectedup to a specific temperature beyond which they where no longer visible(FIG. 2). Addition of Compound 15e resulted in an increased meltingtemperature from ca +55° to +58° C. whereas addition of Wortmanninresulted in an increased melting temperature between ca +55° and +65° C.A comparison with previous data on the melting temperatures ofIMAC-purified PIK3C3 construct with and without the addition of Compound15e or Wortmannin showed good correlation between the two sets ofresults. This experiment shows that it is possible to detect an increasein melting temperature of the PIK3C3 construct in the cellularenvironment after addition and binding of either of two PIK3C3inhibitors Wortmannin and Compound 15e.

Example 3 Determination of Melting Temperature of Four Test Proteins inMammalian Cell Systems

In order to determine the melting temperature of four proteins, lysatewas prepared from cultured mammalian cells and exposed to a panel ofincreasing temperatures. After the temperature steps, precipitatedprotein was removed, leaving only soluble protein (i.e. up to theprotein's specific melting temperature) to be detected.

Materials and Methods

Lysate was prepared from cultured human adenocarcinoma cells (A549).Cells were disrupted on ice in hypotonic buffer and with homogenisation.The suspensions were freeze-thawed multiple times and all insolubleaggregates and cell debris were pelleted by centrifugation aftercompleted lysis. The supernatant containing optically clear cytosolicfraction was aliquoted into 8-strip PCR tubes and subject to a panel ofincreasing temperatures. After heating for three minutes, the sampleswere cooled and precipitated protein was pelleted by centrifugation. Thesupernatant, containing soluble protein, was loaded on separating gels.In addition, the pellet containing precipitated protein from the highesttemperature was dissolved in loading buffer and loaded in the last laneof the gel in order to show the presence of the protein in thisfraction. The gels were blotted onto a Western blot nitrocellulosemembrane. The membrane was washed and blocked with blocking reagent andprobed with primary against dihydrofolate reductase (DHFR), thymidylatesynthase (TS), cyclin dependent kinase-2 (CDK-2) and Protein Kinase C(PKC). Secondary antibodies were bound, and the signal from the boundsecondary antibody was detected by chemiluminescense and recorded with aCCD camera. The intensities were measured and plotted.

Results

The dark bands indicating the presence of soluble protein were detectedup to a specific temperature; they became fainter and ultimatelydisappeared at higher temperatures (FIG. 3). This experiment shows thatthese proteins have a distinct melting temperature and behaviour in thecellular environment and that these melting points can easily bedetected by monitoring the precipitation of the protein (FIG. 4).

Example 4 Detection of an Increase of Melting Temperature after Bindingto a Ligand in Mammalian Cells

In order to investigate the possible increase or decrease in meltingtemperature after addition and binding of inhibitors, the proteinsdihydrofolate reductase (DHFR) and thymidylate synthase (TS) werestudied in cultured mammalian cells.

Lysate from cultured adenocarcinoma cells were treated with one of twoinhibitors; raltitrexed or methotrexate, where the possible stabilisingor destabilising effects of methotrexate was analysed for DHFR andraltitrexed was analysed for TS. After treatment with or without theinhibitors the samples were subjected to a heating step followed byremoval of precipitated protein. The melting temperatures of treatedsamples were compared to those of untreated samples.

Materials and Methods

Lysate was prepared from cultured human adenocarcinoma cells (A549).Cells were disrupted on ice in hypotonic buffer and with homogenisation.The suspensions were freeze-thawed multiple times and all insolubleaggregates and cell debris were pelleted by centrifugation aftercompleted lysis. The supernatant containing optically clear cytosolicfraction was divided into four aliquots, two were supplemented withtheir respective ligand with one corresponding negative control. Theconcentration of added ligand was 10 times the described IC50 value forthe drug/target interaction. Each ligand was dissolved in DMSO and thefinal concentration was set to 1%.

After incubation, each aliquot was divided into 8-tube PCR strips andsubjected to an array of temperatures ranging from +36° C. to 60° C. forDHFR and +51° C. to +69° C. for TS (guided by the melt curve fromExample 3). After heating for three minutes, precipitated protein waspelleted by centrifugation. The resulting supernatants were loaded on aseparating gel and transferred to a Western blot nitrocellulosemembrane. After blocking of the membrane, it was probed with primary andsecondary antibodies. The signal from the bound secondary antibody wasdetected by chemiluminescense and recorded with a CCO camera. Theintensities were plotted to visualize the changes in melting temperaturefollowing ligand treatment.

Results

Addition of methotrexate or raltitrexed resulted in an increased meltingtemperature (FIGS. 5 and 6). This experiment shows that it is possibleto detect an increase in melting temperature of DHFR or TS in thecellular environment after the addition and binding of the respectiveinhibitors methotrexate and raltitrexed.

Example 5 Cellular Thermal Shift Studied in Different Cell Systems andOrganisms to Determine Changes in Melting Temperature Upon Binding of aLigand

In order to study the effects of ligand binding in different systems,the possible stabilising or destabilising effects upon addition of theligand TNP-470, an antiangiogenic agent, to the proteinmethionine-aminopeptidase-2, was determined. Studies were done on cellsfrom two different systems: a) intact cow liver biopsies incubated withTNP-470 and b) human cultured cells incubated with TNP-470. All sampleswere compared to reference samples, which had not been exposed toTNP-470. After treatment with or without the inhibitor, the samples wereprepared and subjected to an array of increasing temperatures. Theprecipitated protein fraction was pelleted by means of centrifugationand the supernatant from each temperature step was analysed on gels andby Western blot. Melting temperatures of proteins treated with TNP-470were compared to those of untreated samples.

Materials and Methods

Lysate was prepared for cultured human cells (K562) and cow liversamples by disruption on ice in hypotonic buffer and withhomogenisation. The suspensions were freeze-thawed multiple times andall insoluble aggregates and cell debris were pelleted by centrifugationafter completed lysis. The lysate of each cell type was divided into twoaliquots, where one was supplemented with TNP-470 (dissolved in pureDMSO) and the other with an equivalent volume of pure DMSO. Afterincubation at room temperature the samples were divided into fractionsof 50 microliters in 8-tube PCR strips and subsequently placed in aVeriti thermocycler.

Next, a series of temperatures were applied to different samples rangingbetween +56° C. to +88° C. with 2 or 4° C. increments and a 3 minutehold at each step. Following heating, the samples were cooled and theprecipitated protein pelleted by centrifugation. 20 microliters of eachsupernatant was removed, supplemented with gel loading buffer and fullydenatured by heating. The samples were loaded on a separating gel, whichafter full run time was blotted onto a nitrocellulose membrane. Themembrane was washed and blocked with blocking reagent and probed withprimary and secondary antibodies. The signal from the bound secondaryantibody was detected by chemiluminescense and recorded with a CCDcamera. The intensities were plotted to visualize the changes in meltingtemperature following ligand treatment.

Results

Dark bands on the Western blot membrane indicates the presence ofsoluble protein still in the supernatant. The soluble protein wasdetected up to a specific temperature beyond which they were no longervisible. Addition of TNP-470 resulted in a shift in melting temperaturefor human cell lysate from 62° C. to 80° C. (an 18° C. shift) and forcow liver lysate from 66° C. to 80° C. (a 14° shift) (FIG. 7).

Example 6 Dose Response Curve from the Concentration Dependence of theThermal Stabilization

For the purpose of constructing a dose-response curve to estimateapparent binding constants, cow liver lysate was subjected to a dilutionseries of ligand TNP-470, specifically targeting methionineaminopeptidase-2. Prior to this Example, curves corresponding to treatedand untreated samples have been obtained (see Example 5) where the dosehas been set at saturating levels. The differences in meltingtemperature can then be used to decide on a temperature where a treatedsample is still present whilst an untreated sample will be precipitated.For this Example the temperature was set to +76° C. The dilution serieswas constructed as a series of 10-fold dilutions. The generated curvegives an indication of the concentration of the ligand needed to engagethe target protein in the lysate.

Materials and Methods

Lysate was prepared from cow liver samples. Cells were disrupted on icein hypotonic buffer and with homogenisation. The suspensions werefreeze-thawed multiple times and all insoluble aggregates and celldebris were pelleted by centrifugation after completed lysis. Thesupernatant containing optically clear cytosolic fraction was aliquotedinto 8-strip PCR tubes where each tube contained an increasing amount ofthe ligand TNP-470 so that the concentration of ligand ranged between 1picomolar and 100 nanomolar and with the DMSO concentration at 1% of thefinal volume. The samples were incubated and subsequently heated to 76°C. for 3 minutes. Following heat treatment the samples were cooled andthe precipitated fraction was pelleted by centrifugation. 20 microliterof each supernatant was removed and supplemented with gel loading bufferand fully denatured by heating. The samples were loaded on a separatinggel, which after full run time was blotted onto a nitrocellulosemembrane. The membrane was washed and blocked with blocking reagent andprobed with primary and secondary antibodies. The signal from the boundsecondary antibody was detected by chemiluminescense and recorded with aCCD camera. The intensities were measured and plotted.

Results

Dark bands on the Western blot membrane indicates presence of protein inthe supernatant. If no or very little protein is present, no or very lowsignal will be visible. As the concentration of ligand increases, theamount of stabilised protein also increases. This is observed as agradually increasing signal of the dark band on the Western blotmembrane. Plotting the integrated intensities will render adose-response curve (FIG. 8), which makes it possible to pinpoint anapparent concentration where half of the protein in the sample will havebeen engaged by a bound ligand (i.e. stabilised). This can have usefulapplications to set dosing regimes for patients or to find a therapeuticwindow for a drug by studies of apparent binding constants in differentorgans of the body.

Example 7 Biosensor Application—Measurement of Presence of a Ligand inComplex Fluids

The presence of a ligand (e.g. a drug) for which there is a cognateligand binding protein (e.g. a drug target) can be indicated even incomplex test samples lacking the target protein. This is achieved byadding an aliquot of a sample containing the protein (e.g. lysate of thetarget cell or a purified protein) to the biological fluid test sample.In line with Example 6, a dose response curve can also be constructedusing serial dilutions of a biological fluid (e.g. blood plasma orserum) containing the ligand of interest. The curve thus created can befitted on to a dose-response curve generated by spiking to give anestimated concentration of the ligand in the biological fluid.

Materials and Methods

Lysate was prepared from cultured human adenocarcinoma cells (A549).Cells were disrupted on ice in hypotonic buffer and with homogenisation.The suspensions were freeze-thawed multiple times and all insolubleaggregates and cell debris were pelleted by centrifugation aftercompleted lysis. The supernatant containing optically clear cytosolicfraction was aliquoted into 8-strip PCR tubes where each tube containedan increasing amount of the ligand TNP-470 dissolved in heat treatedA549 lysate (heat treatment at 76° C. precipitated all target protein(methionine aminopeptidase-2) and ensured that no ligand would beconsumed). As in Example 6, the concentrations ranged between 1picomolar and 100 nanomolar effective concentration.

The samples where incubated and subsequently heated to 76° C. for 3minutes. Following heat treatment the samples were cooled and theprecipitated fraction was pelleted by centrifugation. 20 microliter ofeach supernatant was removed and supplemented with gel loading bufferand fully denatured by heating. The samples were loaded on a separatinggel, which after full run time was blotted onto a nitrocellulosemembrane. The membrane was washed and blocked with blocking reagent andprobed with primary and secondary antibodies. The signal from the boundsecondary antibody was detected by chemiluminescense and recorded with aCCO camera. The intensities were measured and plotted.

Results

The heat-treated lysate was generated to mimic a biological fluiddeficient in target protein. The spiking of the heat-treated lysate withligand and the serial dilution thereof then produced a response curve(FIG. 9) that could be compared to and fitted on to an in vitrogenerated dose-response curve to get an estimate of how much ligand ispresent in the sample.

Example 8 Ligands Targeting Specific Protein Variants

Within a human population, proteins exist as different variants, usuallywith a small number of amino acid substitutions. In some instances thesesubstitutions promote diseases, such as, for example, cancer. Theprotein B-raf is involved in pathways where disturbances in regulationor function can cause such diseases. Many different amino acidsubstitutions have been described for B-raf that result in an oncogenicprotein. Amino acid substitutions can also make a protein less capableof binding drugs, which is one driving cause behind resistancedevelopment during cancer treatment.

The ligand SB590885 is known to bind the V600E variant of B-raf, whichcan be hard to treat with medication such as Sorafenib. In this Example,we show that there is a difference in stability in the substitutedversus the wild type protein and that the binding of ligand affects theprotein variants to a different extent.

Materials and Methods

Lysate was prepared from cultured human A375 cells containing the V600Esubstitution in B-raf and K562, containing the wild type versionthereof. Cells were disrupted on ice in hypotonic buffer and withhomogenisation. The suspensions were freeze-thawed multiple times andall insoluble aggregates and cell debris were pelleted by centrifugationafter completed lysis. The supernatants containing optically clearcytosolic fraction were each aliquoted into two tubes where each tubecontained either the ligand SB590885 dissolved in DMSO or pure DMSO forcontrol. The samples where incubated and subsequently aliquoted into8-tube PCR strips in fractions of 50 microliters. A series oftemperatures were applied to the different samples ranging between +44°C. to +62° C. with 2° C. increments and a 3 minute hold at eachtemperature. Following heating, the samples were cooled and theprecipitated protein pelleted by centrifugation. 20 microliter of eachsupernatant was removed and supplemented with gel loading buffer andfully denatured by heating. The samples were loaded on a separating gel,which after full run time was blotted onto a nitrocellulose membrane.The membrane was washed and blocked with blocking reagent and probedwith primary and secondary antibodies. The signal from the boundsecondary antibody was detected by chemiluminescense and recorded with aCCO camera. The intensities were normalized and plotted to visualize thechanges in melting temperature following ligand treatment (FIG. 10).

Results

The melting curves in FIG. 10 show that substituted V600E B-raf is lessstable than wild type if no stabilising ligand is present. Upontreatment, V600E substituted B-raf is stabilised with approximately a 6°C. increase in the melting temperature, while the wild type protein oncestabilised only showed a 3° C. increase in the melting temperature.After stabilisation, both the V600E B-raf and the wild-type B-raf showeda melting temperature of 55° C.

The invention claimed is:
 1. A method for identifying a ligand capableof binding to a target protein comprising the steps of: a) exposing anon-purified sample comprising the target protein and a test molecule toa temperature which is capable of causing or enhancing precipitation ofthe unbound target protein to a greater extent than it is capable ofcausing or enhancing precipitation of the target protein bound to theligand, wherein the target protein in said non-purified sample iscomprised within or on cells; b) subjecting said sample to conditionscapable of causing cell lysis; c) separating soluble from insolubleprotein in the product of step b); and d) analysing the soluble proteinfractions of step c) and optionally the insoluble protein fraction ofstep c) for the presence of the target protein, wherein the targetprotein is detected by affinity binding to a detection moiety or by massspectrometry, and wherein the test molecule is identified as a ligandcapable of binding to the target protein if the level of target proteinin said soluble fraction is higher than the level of target proteindetected in a soluble fraction obtained in a control reaction.
 2. Themethod of claim 1, wherein a non-purified sample comprising the targetprotein without added test molecule is also subjected to the method ofclaim 1 in a control reaction.
 3. The method of claim 1, wherein thenon-purified sample is a cell colony, a liquid culture of cells or apatient or animal sample.
 4. The method of claim 3, wherein the patientor animal sample is obtained directly from the patient or animal and/oris a tissue sample.
 5. The method of claim 4, wherein the tissue sampleis blood, serum, plasma or lymph.
 6. The method of claim 1, wherein theligand is a protein, DNA molecule, RNA molecule, a cellular metabolite,a drug or another chemical.
 7. The method of claim 1, wherein the targetprotein is identified using antibodies.
 8. The method of claim 1,wherein the separating step c) is a step of centrifugation, filtrationor affinity separation.
 9. The method of claim 8, wherein the targetprotein in the filtrate from the filtration step is captured on a solidsupport prior to the analysis step d).
 10. The method of claim 1,wherein the non-purified sample is a cell colony, separation step c) isfiltration and wherein said cell colony is lifted on a filter and lysisis carried out directly on the colony on the filter.
 11. A method foridentifying a ligand capable of binding to a target protein comprisingthe steps of: a) exposing a non-purified sample comprising the targetprotein and a test molecule to a temperature which is capable of causingor enhancing precipitation of the target protein bound to the ligand toa greater extent than it is capable of causing or enhancingprecipitation of the unbound target protein, wherein the target proteinin said non-purified sample is comprised within or on cells; b)subjecting said sample to conditions capable of causing cell lysis; c)separating soluble from insoluble protein in the product of step b); andd) analysing the soluble protein fraction of step c) and optionally theinsoluble protein fraction of step c) for the presence of the targetprotein, wherein the target protein is detected by affinity binding to adetection moiety or by mass spectrometry, and wherein the test moleculeis identified as a ligand capable of binding to the target protein ifthe level of target protein in said soluble fraction is lower than thelevel of target protein detected in a soluble fraction obtained in acontrol reaction.
 12. The method of claim 11, wherein a non-purifiedsample comprising the target protein without added test molecule is alsosubjected to the method of claim 1 in a control reaction.
 13. The methodof claim 11, wherein the non-purified sample is a cell colony, a liquidculture of cells or a patient or animal sample.
 14. The method of claim13, wherein the patient or animal sample is obtained directly from thepatient or animal and/or is a tissue sample.
 15. The method of claim 14,wherein the tissue sample is blood, serum, plasma or lymph.
 16. Themethod of claim 11, wherein the ligand is a protein, DNA molecule, RNAmolecule, a cellular metabolite, a drug or another chemical.
 17. Themethod of claim 11, wherein the target protein is identified usingantibodies.
 18. The method of claim 11, wherein the separating step c)is a step of centrifugation, filtration or affinity separation.
 19. Themethod of claim 18, wherein the target protein in the filtrate from thefiltration step is captured on a solid support prior to the analysisstep d).
 20. The method of claim 11, wherein the non-purified sample isa cell colony, separation step c) is filtration and wherein said cellcolony is lifted on a filter and lysis is carried out directly on thecolony on the filter.